Kushiyev et al. Egyptian Journal of Biological (2018) 28:55 Egyptian Journal of https://doi.org/10.1186/s41938-018-0062-z

RESEARCH Open Access Efficacy of native entomopathogenic , fumosorosea, against bark and ambrosia , Anisandrus dispar Fabricius and Xylosandrus germanus Blandford (Coleoptera: Curculionidae: Scolytinae) Rahman Kushiyev, Celal Tuncer, Ismail Erper* , Ismail Oguz Ozdemir and Islam Saruhan

Abstract The efficacy of the native , TR-78-3, was evaluated against females of the bark and ambrosia beetles, Anisandrus dispar Fabricius and Xylosandrus germanus Blandford (Coleoptera: Curculionidae: Scolytinae), under laboratory conditions by two different methods as direct and indirect treatments. In the first method, conidial suspensions (1 × 106 and 1 × 108 conidia ml−1) of the fungus were directly applied to the beetles in Petri dishes (2 ml per dish), using a Potter spray tower. In the second method, the same conidial suspensions were applied 8 −1 on a sterile hazelnut branch placed in the Petri dishes. The LT50 and LT90 values of 1 × 10 conidia ml were 4.78 and 5.94/days, for A. dispar in the direct application method, while they were 4.76 and 6.49/days in the branch application 8 −1 method. Similarly, LT50 and LT90 values of 1 × 10 conidia ml for X. germanus were 4.18 and 5.62/days, and 5.11 and 7.89/days, for the direct and branch application methods, respectively. The efficiency of 1 × 106 conidia ml−1 was lower than that of 1 × 108 against the beetles in both application methods. This study indicates that I. fumosorosea TR-78-3 had a significant potential as a biological control agent against A. dispar and X. germanus. Further studies are necessary to evaluate the efficacy of the isolate on the pests under field conditions. Keywords: Bark and ambrosia beetles, Biological control, Entomopathogenic fungus, Isaria fumosorosea, Hazelnut

Background fungi are carried by females in a specialized structure Ambrosia beetles (Coleoptera: Curculionidae), having known as mycangium or mycangia (Six 2003). More- approximately 3400 species in the sub-families Scolytinae over, ambrosia beetles often inoculate secondary pathogenic and Platypodinae, are among the major pests which threat fungi such as Fusarium spp. (Kessler 1974) or bacteria (Hall many fruit and forest trees (Hulcr and Dunn 2011). These et al. 1982) during entry into trees (Oliver and Mannion beetles make galleries through sapwood (xylem) of host 2001). Consequently, these beetles could harm the trees by trees and cultivate symbiotic fungi such as Ambrosiella carrying plant diseases, tunneling the trees, and farming spp. and Raffaelea spp. in the galleries for their food symbiotic fungi. Among bark and ambrosia beetles, (Harrington 2005). The symbiotic fungi which have Anisandrus dispar Fabricius and Xylosandrus germanus co-evolved with ambrosia beetles provide nutrition required Blandford (Coleoptera: Curculionidae: Scolytinae) are for the development of larvae and adults (Norris 1979). The the most prevalent pest species all over the world (Oliver and Mannion 2001; Rabaglia et al. 2006; Ranger et al. 2016;Ak2016). These pests are polyphagous and damage * Correspondence: [email protected] Faculty of Agriculture, Department of Plant Protection, Ondokuz Mayis many perennial plants, including hazelnut (Bucini et al. University, 55139 Atakum, Samsun, Turkey 2005 and Ak et al. 2011).

© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Kushiyev et al. Egyptian Journal of Biological Pest Control (2018) 28:55 Page 2 of 6

A. dispar, X. germanus,andXyleborinus saxesenii kept in plastic boxes (20 × 25 × 40 cm), and directly Ratzeburg are among the significant pests of hazelnut transferred to the laboratory (Ondokuz MayısUniversity, in Turkey (Ak 2016;Tunceretal.2017). These beetles Agriculture Faculty, Plant Protection Department, Samsun, cause crop losses by draining hazelnut branches and Turkey). The branches were dissected by pruning scissors. trees, especially in orchards at coastline of the Black Only females were collected from the galleries and Searegioninthecountry,whichhashighgroundwater inspected under Leica EZ4 stereomicroscope at × 40–70 level (Saruhan and Akyol 2012). magnification to separate healthy adults of A. dispar and Ambrosia beetles are difficult to control as their majority X. germanus for use in bioassays. As in many EPF studies of life is spent under the bark of host trees (Reding et al. (Castrillo et al. 2015), only females were used in the 2010). Therefore, can be ineffective against present study. The males are rare in the population of these beetles unless applied repeatedly or application ambrosia beetles (about 10:1 in favor of female), flightless, timing coincides with flight time of the beetles (Oliver and rarely seen outside the gallery (Ranger et al. 2016). and Mannion 2001). Keeping in view these facts, effective and eco-friendly alternative control methods are inevitable Fungal isolate in the country. Considering environmental conditions, use EPF isolate TR-78-3 used in the present study was of entomopathogenic fungi (EPF) against A. dispar and isolated from the pupae of Hyphantria cunea (Lepidoptera: X. germanus could be an alternative pest management Erebidae) commonly called fall webworm, collected from approach in hazelnut orchards. The Black Sea region hazelnut orchards (Samsun, Turkey). Single- isolate receives frequent rainfall and has high humidity and was obtained by serial dilution (Dhingra and Sinclair 1995) low temperatures per year, and these environmental and identified as I. fumosorosea by Dr. Richard A. Humber, conditions are ideal for the development of entomopatho- USDA-ARS Biological Integrated Pest Management Unit. genic fungi (Erper et al. 2016). The fungus was prepared and stored at 4 °C on Sabouraud EPF such as (Bals.) Vuill., Lecani- dextrose agar (SDA; Merck Ltd., Darmstadt, Germany) cillium spp., anisopliae (Metch) Sorok, and slants and also in cryogenic tubes containing 15% glycerol Isaria fumosorosea Wize are used to control several at − 80 °C. The isolate was deposited in the fungal culture pests around the world (Zimmermann 2007a, 2007b, 2008; collection of the Mycology Laboratory, Ondokuz Gurulingappa et al. 2011). Among those, I. fumosorosea was Mayıs University, Agriculture Faculty, Plant Protection known as fumosoroseus since 30 years, and Department, Samsun, Turkey, and in the USDA-ARS then transferred to the genus Isaria (Zimmermann 2008). Entomopathogenic Fungal Culture Collection in Ithaca, EPF are important control agents of ambrosia beetles, NY (ARSEF 12173). as they may affect larvae in the galleries as well as adults outside the host trees (Castrillo et al. 2013). Some EPF Inoculum of I. fumosorosea have been found associated with ambrosia and bark EPF, I. fumosorosea isolate TR-78-3 was plated on SDA beetles (Popa et al. 2011). Several studies have indicated and incubated (Binder KBWF 240; Germany) at 25 °C for that B. bassiana, M. anisopliae,andI. fumosorosea were 15 days. Conidia were harvested by sterile distilled water effective against ambrosia beetles such as Trypodendron containing 0.02% Tween 20. Mycelia were removed by lineatum Olivier, X. germanus, Xylosandrus crassiusculus filtering conidia suspensions through four layers of sterile (Motschulsky), and Xyloborus glabratus Eichhoff (Castrillo cheese cloth. The suspensions of conidia were adjusted − et al. 2011, 2013 and 2015). Castrillo et al. (2011)found to 1 × 106 and 1 × 108 conidia ml 1,usingaNeubauer that commercial strains of B. bassiana and M. anisopliae hemocytometer under Olympus CX31 compound micro- induced high mortality in females of X. germanus,as100% scope (Olympus America Inc., Lake Success, NY) (Saruhan of their offspring larvae was infected. et al. 2015). The present study shed a light on the efficacy of two − conidial concentrations (1 × 106 and 1 × 108 conidia ml 1) Conidial germination assessment of I. fumosorosea TR-78-3 against females of A. dispar and The viability of conidia of the isolate was determined. A − X. germanus, using two different application methods conidial suspension was adjusted to 1 × 104 conidia ml 1, under laboratory conditions. and 0.2 ml was sprayed onto Petri dishes (9 cm diam- eter), containing potato dextrose agar (PDA; Merck Ltd., Material and methods Darmstadt, Germany). The Petri dishes were incubated cultures at 25 ± 1 °C. After 24 h of incubation, percentage of ger- Hazelnut orchards in Terme district of Samsun province, minated conidia was determined, using Olympus CX-31 Turkey, were surveyed to collect hazelnut branches infested compound microscope at × 400 magnification. Conidia with A. dispar and X. germanus during March–April 2017. were regarded as germinated when they produced a The infested branches were cut into 30-cm-long pieces, germ tube at least half of the conidial length (Saruhan et Kushiyev et al. Egyptian Journal of Biological Pest Control (2018) 28:55 Page 3 of 6

al. 2015). The germination ratios of the fungus were de- on the cadavers under Leica EZ4 stereomicroscope termined by examining a minimum of 200 conidia from (Kocaçevik et al. 2016). each of three replicate dishes. Statistical analysis Bioassays Abbott correction was not used since mortality was Females of A. dispar and X. germanus were surface disin- lower more than 5% in the control. Independent time- fected with 70% ethanol for 10 s and placed on an auto- mortality data from bioassays were analyzed by Probit claved filter paper for drying (Castrillo et al. 2013). The analysis program (POLO-PLUS Ver.2.0) to calculate 50% hazelnut branches (5 cm length and 1 cm diameter), used (LT50) and 90% (LT90) lethal time. In lethal time analysis, in the bioassays, were sterilized in autoclavable polyethylene Log-Probit analysis calculations were considered. Slopes bags (30 × 30 cm) for 1 h at 121 °C on two successive days. of regression lines were compared to each other by their The Petri dishes that were lined with the moist sterile filter standard errors. LT50 and LT90 values of two application paper at the bottom were used in all experiments. The methods and two conidial suspensions were compared bioassays were carried out by using two different methods; based on overlapping of 95% confidence intervals. the first, two conidial suspensions (1 × 106 and 1 × 108 − conidia ml 1) of the isolate were directly sprayed to Results and discussion females of A. dispar and X. germanus in the Petri dishes Conidia viability of I. fumosorosea isolate TR-78-3 was (2 ml per dish), using a Potter spray tower (Burkard, assessed before bioassays, and approximately (100%) 6 Rickmansworth, Hertz UK), and a sterilized hazelnut germination was obtained. The LT50 values of 1 × 10 − branch was placed in each Petri dish, and the second conidia ml 1 for A. dispar were 6.92 and 6.66 days for method, same amount of conidial suspensions of the branch and insect application, respectively. Correspondent fungus was applied to Petri dishes containing a sterilized LT90 values, for the same concentration, were 11.16 and 8 −1 hazelnut branch with same method, and then the sterile 10.57 days. The LT50 values of 1 × 10 conidia ml were females were released in the dishes. Control Petri dishes 4.76 and 4.78 days, while the LT90 values were 6.49 and were treated by sterile distilled water (2 ml) containing 5.94 days for branch and insect application methods, 0.02% Tween 20. The spray tower was disinfected by 70% respectively (Table 1). The isolate tested against A. dispar ethanol and sterile distilled water after each application. resulted in increasing mortality rate, starting from day 4 in All Petri dishes were loosely covered by Parafilm and incu- all application methods, with both tested concentrations. − bated at 25 ± 1 °C and 75 ± 5% RH, 16:8 h light to dark The efficacy of 1 × 108 conidia ml 1 on A. dispar increased period in a Binder incubator (Model KBWF 240; rapidly; by days 6 and 8, the mortality rates attained 75–90 Germany). The experiment had five replications (a Petri and 100% in both methods, respectively. Nevertheless, the − dish each), by placing five in each dish. Different efficacy of 1 × 106 conidia ml 1 of the isolate was lower − groups of insects were used for 8 successive days, provided than that of 1 × 108 conidia ml 1 (Fig. 1). In addition, all independence of the observations on mortality on over time applications of I. fumosorosea caused (100%) mycosis on (Robertson et al. 2007). The dead adults were removed daily dead females of A. dispar. Kushiyev et al. (2017)estimated and immediately surface disinfected by dipping in 1% the LT50 and LT90 values of M. anisopliae TR-106 isolate − sodium hypochlorite (NaOCl) for 3 min and in 70% ethanol at 1 × 108 conidia ml 1 concentration on females of A. for 3 min. Then, the dead insects were washed three times dispar as 4.03 and 5.39 days at branch application and in sterile distilled water and placed in a moisture chamber. 3.67 and 4.22 days at insect application, respectively. Mortality rates were confirmed by examination of hyphae Additionally, M. anisopliae TR-106 isolate applied with

Table 1 Probit analysis data on mortality time of Anisandrus dispar females after applications of two conidial concentrations of Isaria fumosorosea isolate TR-78-3 on branch and insect 1×106 conidia ml−1 1×108 conidia ml−1 On branch On insect On branch On insect

LT50 (95% CI) 6.92 (6.27–7.98) a* 6.66 (6.06–7.55) a 4.76 (4.37–5.14) b 4.78 (4.44–5.09) b

LT90 (95% CI) 11.16 (9.22–17.12) a 10.57 (8.88–15.36) a 6.49 (5.91–7.60) b 5.94 (5.51–6.76) b Slope ± SE 10.58 ± 2.21 b 10.96 ± 2.20 b 16.33 ± 2.72 a 23.23 ± 4.25 a χ2 21.07 6.78 13.14 7.92 Df 30 30 30 30 Heterogeneity 0.70 0.23 0.44 0.26 *Within rows, means followed by same lower case letter do not differ significantly Kushiyev et al. Egyptian Journal of Biological Pest Control (2018) 28:55 Page 4 of 6

8 −1 that LT50 and LT90 values of 1 × 10 conidia ml of B. bassiana TR-217 isolate against female adults of X. germa- nus were 5.96 and 11.79 days for branch method and 6.03 and 10.80 days for direct insect spray method. As a result, the LT50 and LT90 values in the present study decreased with increasing conidial concentration of the isolate at all applications. However, comparisons of the confidence intervals indicated insignificant differ- ences between LT90 values at both concentrations against females of X. germanus in branch application method. In addition, the isolate caused (100%) mortality − rate with (1 × 108 conidia ml 1) in both methods by day Fig. 1 Mortality rates of Anisandrus dispar treated with Isaria 8 after inoculation against females of A. dispar. Same fumosorosea TR-78-3 at different methods and conidial concentrations. concentration of the isolate resulted to 90 and 100% – – a1×106 conidia ml 1 on branch, b 1 × 106 conidia ml 1 on insect, c mortality rate of X. germanus by day 8, in the branch 8 –1 8 –1 1×10 conidia ml on branch, d 1 × 10 conidia ml on insect and insect application methods, respectively. Some researchers have reported that the efficacy of EPF increases at high concentrations, which shorten the time two methods against the caused 100% mortality required for insect mortality (Kocaçevik et al. 2016). and about 95% mycosis after 8 days. Similarly, Tuncer et al. (2016) and Kushiyev et al. (2017) 6 −1 The LT50 values of (1 × 10 conidia ml ) the isolate found that LT50 and LT90 values of M. anisopliae TR-106 applied to X. germanus were 7.81 and 7.02 days in and B. bassiana TR-217 isolates applied to females of A. branch and insect application methods, respectively. dispar and X. germanus decreased by increasing conidial Correspondent LT90 values were 11.03 and 11.46 days. concentration. The present study also indicated that all 8 −1 The LT50 and LT90 values of 1 × 10 conidia ml of the applications of I. fumosorosea caused 100 and 95–100% isolate were 5.11 and 7.89 days for branch application mycosis on dead females of A. dispar and X. germanus, method, while they were 4.18 and 5.62 days for insect respectively. Nevertheless, Demir et al. (2013)statedthat application method (Table 2). The tested isolate caused I. fumosorosea isolate caused mortality to Plagiodera versi- mortality against X. germanus by day 4 in all treatments, colora Laicharting (Chrysomelidae), but did not produce − except the 1 × 106 conidia ml 1 at the branch application mycosis on dead cadavers. − method. High concentration (1 × 108 conidia ml 1), using In the present study, significant difference was insect application method caused 40% mortality rate by observed between the two application methods for 1 × − day 4, reaching 100% by day 7. Likewise in the branch 108 conidia ml 1 applied to X. germanus;however,there application method, an increasing mortality rate was ob- was no difference between the methods for all other served by day 4, raised to 70% by day 6 and 90% by day 8. applications (P > 0.05). Castrillo et al. (2015)reported Conversely, efficacy of the low concentration (1 × 106 co- insignificant differences in the mortality rates of X. − nidia ml 1) remained lower than that of the high concen- glabratus females, exposed to B. bassiana strain (GHA) tration of the isolate (Fig. 2). Moreover, I. fumosorosea and I. fumosorosea strains (Ifr 3581 and PFR), by dipping TR-78-3 isolate caused 95–100% mycosis on dead females in fungal suspension or placing on treated avocado of X. germanus. Similarly, Tuncer et al. (2016)reported branches. Tuncer et al. (2016) investigated the efficiency

Table 2 Probit analysis data on mortality time of Xylosandrus germanus females after applications of two conidial concentrations of the Isaria fumosorosea isolate TR-78-3 on branch and insect 1×106 conidia ml−1 1×108 conidia ml−1 On branch On insect On branch On insect

LT50 (95% CI) 7.81(7.16–9.27)a* 7.02(6.34–8.18)a 5.11(4.63–5.60)b 4.18(3.82–4.52)c

LT90 (95% CI) 11.03(9.28–17.83)a 11.46(9.38–18.10)a 7.89(6.95–9.85)a 5.62(5.12–6.58)b Slope ± SE 14.66 ± 3.70 ab 10.32 ± 2.19 b 11.65 ± 1.93 b 17.12 ± 2.95 a χ2 5.12 11.94 14.92 9.02 Df 30 30 30 29 Heterogeneity 0.17 0.40 0.50 0.31 *Within rows, means followed by same lower case letter do not differ significantly Kushiyev et al. Egyptian Journal of Biological Pest Control (2018) 28:55 Page 5 of 6

environmental conditions to use EPF in biocontrol of A. dispar and X. germanus, because it is rainy and humid and has low annual temperatures (Erper et al. 2016).

Conclusions The present study showed that the EPF, I. fumosorosea isolate TR-78-3, seemed to be a promising biological control agent against A. dispar and X. germanus. However, further studies are necessary to evaluate the efficacy of the isolate on the pests under field conditions.

Acknowledgements The authors extend thanks to Dr. Richard A. Humber for his kind help with the morphological characterizations of the entomopathogenic fungus. Rahman Kushiyev is thankful to the Scientific and Technological Council of Fig. 2 Mortality rates of Xylosandrus germanus treated with Isaria Turkey (TUBITAK) for providing fellowship under 2215 Graduate Scholarship fumosorosea TR-78-3 at different methods and conidial concentrations. program for international students. a1×106 conidia ml–1 on branch, b 1 × 106 conidia ml–1 on insect, c 8 –1 8 –1 1×10 conidia ml on branch, d 1 × 10 conidia ml on insect Availability of data and materials All data are available at the end of the article, and the materials used in this work are of high quality and grade. on insect and branch treatments of M. anisopliae Authors’ contributions TR-106 and B. bassiana TR-217 isolates at 1 × 106 and IE, RK, CT, IOO, and IS designed the study, supervised the work, and wrote 8 −1 the manuscript with input from all authors. IE, RK, IOO, and IS carried out the 1×10 conidia ml against females of X. germanus and experiments. CT analyzed the data. All authors read and approved the final found insignificant differences between application methods manuscript. and LT50 and LT90 values. In another study, Castrillo et al. (2013)demonstratedthatB. bassiana (GHA and Naturalis) Ethics approval and consent to participate Not applicable. and M. anisopliae strain (F52) applied to females of X. crassiusculus caused 77, 96, and 79% mortality rates, Consent for publication respectively 5 days after treatment. Moreover, they stated Not applicable. that the females exposed to beech stems treated with same Competing interests 7 −1 entomopathogen strains at 1 × 10 conidia ml had lower The authors declare that they have no competing interests. survival rates. EPF can be effective when applied directly on the Publisher’sNote insect or when the insect contacts the applied surface. Springer Nature remains neutral with regard to jurisdictional claims in Moreover, they provided a good potential control for published maps and institutional affiliations. some pests living in wood of trees, such as ambrosia and Received: 18 April 2018 Accepted: 27 June 2018 bark beetles, as they could also be transported into the gallery by infected individuals (Castrillo et al. 2013). References Castrillo et al. (2011) determined that treated females of Ak K (2016) Comparison of sticky and non-sticky traps against harmful shothole X. germanus with commercial strains of B. bassiana borers (Coleoptera: Curculionidae: Scolytinae) in hazelnut orchards. Anadolu J (Naturalis and GHA) and M. anisopliae (F52) caused Agr Sci 31(1):165–170 Ak K, Saruhan I, Tuncer C, Akyol H, Kiliç A (2011) Ambrosia beetles (Coleoptera: significantly higher mortality rates compared to control, Scolytidae) species and their damage ratios in Kiwi orchards of Ordu and reduced gallery formation and brood production in Province. Turk bull Entomol 1(4):229–234 rearing tubes. They also stated that some treated females Bucini D, Balestra GM, Pucci C, Paparatii B, Speranza S, Zolla CP, Varvaro L (2005) Bio-ethology of Anisandrus dispar F. and its possible involvement in Dieback had 100% infected broods. Treated male adults transferred (Moria) diseases of hazelnut (Corylus avelana L.) plants in Central Italy. Acta fungal inocula to untreated females which reduced their Hortic 686:435–443 breeding activity by 20% (Prazak 1991). In summary, EPF Castrillo D, Dunlap CA, Avery PB, Navarrete J, Duncan RE, Jackson MA, Behle RW, Cave RD, Crane J, Rooney AP, Peña JE (2015) Entomopathogenic fungi as biological did not only cause mortality of adult beetles outside in the control agents for the vector of the laurel wilt disease, the red bay ambrosia host trees but also decreased beetles’ population through beetle, Xyleborus glabratus (Coleoptera: Curculionidae). Biol Control 81:44–50 infecting next-generation broods in the galleries. Castrillo LA, Griggs MH, Ranger CM, Reding ME, Vandenberg JD (2011) Virulence of commercial strains of Beauveria bassiana and Sevim et al. (2013) indicated that EPF were more likely (: ) against adult Xylosandrus germanus (Coleoptera: to have ecological compatibility with pests due to their Curculionidae) and impact on brood. Biol Control 58:121–126 geographical locations and habitats. In addition, The Castrillo LA, Griggs MH, Vandenberg JD (2013) Granulate ambrosia beetle, Xylosandrus crassiusculus (Coleoptera: Curculionidae), survival and brood Middle and Eastern Black Sea region, which has largest production following exposure to entomopathogenic and mycoparasitic hazelnuts growing area of Turkey, has favorable fungi. Biol Control 67:220–226 Kushiyev et al. Egyptian Journal of Biological Pest Control (2018) 28:55 Page 6 of 6

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